Lecture 12 - Lymphocyte Activation Flashcards
Describe the structure of the TCR complex
TCR (α & β chains)
CD3: ε-, δ-, γ-chains ITAMs
What is important about the structure of an ITAM?
Where are they found?
Tryosine residues (which can be phosphorylated) With several other amino acids
They are found in both:
• TCR
• BCR
What happens to the tyrosines in the ITAMs?
Phosphorylated by Src family tyrosine kinases:
• Lck
• Fyn
What regulates the Src family kinases?
Describe this
Inhibition:
1. Csk (C-terminal src kinase)
• A kinase
• Phosphorylates the tyrosine residues on Src family kinase
→ blocks the action of Src family kinase
Activation: 2. CD45 • a phosphatase • removes the phosphates from the Y residues that the Csk put on → activates Src family kinase function
What is the functional relationship between Csk and CD45?
Direct antagonists of each other
What is the structure of a Src family kinase?
Outline the function of each of the domains
SH3 domain
• interacts with the Guanine nucleotide exchange factor
SH2 domain
• docks to other phospho-tyrosines on the receptor
Kinase domain
• tyrosine residues
Describe the conformation of Src-family kinases when activated and inactivated
Activated:
• phosphorylation of activating Tyr on kinase domain
• SH2 domain not bound by kinase domain
Deactivated:
• phosphorylation of inhibitory Tyr on kinase domain
• Kinase domain binds to SH2 domain, inactivating it
Describe the proximal signalling events that occur after MHC-TCR engagement
- MHC binds to TCR
- Recruitment of Lck with the co-receptors (CD4/CD8) to the TCR/CD3 complex
- Lck phosphorylates the Y-residues of ITAMs on CD3
(these Y residues are now a docking site for ZAP-70) - Recruitment of ZAP-70 to phosphorylated tyrosines of the ITAMs
- Phosphoyrlation of ZAP-70 by Lck
- ZAP-70 goes on to phosphorylate LAT and then PLC-γ
What is ZAP-70?
Describe its function
- Syk family kinase
- Activated by Lck (i.e. phosphorylated)
- Phosphorylates LAT & PLC-γ
What is LAT?
Describe the important structural features
Linker of T cell activation
• Membrane bound protein
- Contains tyrosine residues (can be phosphorylated by ZAP-70)
- Phosphorylated tyrosines serve as docking sites for adaptors w/ SH2 domains
Describe the function of LAT
Proximal signalling events
1. Phosphorylation of LAT by ZAP-70
→ activating two different pathways:
a. MAP Kinase pathway
b. PLC-γ pathway
What is PLC-γ?
Describe its basic function
Phospholipase C-γ
• A cytosolic enzyme
• Hydrolyses membrane phospholipids
Describe the two pathways of PLC-γ activity
- Activated PLC-γ
- Production of PIP2
3a. Production of IP3
4a. IP3 increases intracellular Ca2+
5a. Ca2+ activates calcineurin (by binding to Calmodulin)
6a. Calcineurin activates NFAT through phosphatase action
3b. Induction of DAG
4b. DAG and Ca2+ activate PKC
5b. Increase in NF-κB in the cytosol
What does NF-κB and NFAT stand for?
Nuclear factor kappa B
NFAT: nuclear factor of activated T cells
Describe the regulation of NF-κB
- IκB bound to NF-κB, keeping it in the cytoplasm and inactivated
- Phosphorylation of IκB by IκB kinase, in conjunction with PKC
- Proteolysis of IκB in proteasome
- NF-κB moves into the nucleus and binds promoters
- Transcription of interleukins
What is PKC?
Protein kinase C
What is the general function of calcineurin?
Phosphatase - removes phosphate from NFAT
Why are NFAT and NF-κB regulated by location?
They are stuck in the cytoplasm (sequestered from the nucleus) due to:
• NFAT: phosphate
• NF-κB: IκB
Which is the function of Cyclosporine?
Immunosuppressant
• Binds to calcineurin, blocking phosphatase activation
• NFAT is no longer activated by calcineurin
Describe the MAP kinase pathway
- Phosphorylated LAT
- Grb-2 docks to phosphotyrosines on LAT via SH-2 domain
- SOS (Guanine nucleotide exchange factor) docks to SH3 of Grb-2
- Guanine nucleotide exchange factor catalyses
Ras-GDP → Ras-GTP - Ras activates Raf
- Mek
- Erk
- Elk (a transcription factor)
- Elk translocated to the nucleus, and activates another transcription factor (AP-1)
- Gene transcription
What are some Guanine nucleotide exchange factors in the MAPK pathway?
How is it activated?
What is its function?
Sos
• Binds to SH3 domain of the Src family kinase (e.g. Grb-2), which was activated by the receptor
Function
• Replaces GDP with GTP in Ras, activating it
What are the functional responses after T cells activation?
- Cytokine release
- Proliferation
- Differentiation (Memory, effectors)
What drives cytokine release following T cell activation?
- TCR-MHC engagement
- Biochemical pathway
- Transcription factor activation
- Cytokine expression
What are the main transcription factors required for cytokine expression?
How do they function?
- NF-B
- NFAT
- AP-1
The TFs bind to promoters of the genes for the cytokines, ‘turning on’ the gene
Describe the variability of the TCR/CD3 complex
TCR: variable; cell-specific recognition domains
CD3: invariant signalling domains
What does ITAM stand for?
Intracellular tyrosine-based activation motif
What are some Src family kinases?
- Lck
* Fyn
What are the co-receptors that are involved in proximal signalling?
- CD4
* CD8
Where is Lck located?
Associated with the co-receptors in the cell membrane
NB Co-receptors are CD4/CD8
What are Syk family kinases?
Give an important example of one.
Describe their structure
Another family of kinases (distinct form Src)
• e.g. ZAP-70
Structure:
• Tandem SH2 domains (2 x SH2)
• kinase domain
How is ZAP-70 activated?
It is inactive until is interacts with phospho-tyrosines on other proteins (such as CD3 ITAMs)
Describe the events after proximal signalling
At the end of proximal signalling, we have phosphorylated (an thus active) ZAP-70.
- ZAP-70 phosphorylates LAT
Describe the interaction between LAT and PLC-γ
- LAT has been phosphorylated by ZAP-70
- PLC-γ docks on the phosphotyrosines on LAT
- ZAP-70 and Itk phosphorylate PLC-γ
What is PIP2?
What is IP3?
- Phosphatidyl inositol bisphosphate
* Inositol triphosphate
What are the small G proteins in the MAPK pathway?
Ras (or Rac)
What is MAP kinase kinase kinase?
Raf
What is MAP kinase kinase?
Mek
What is MAP Kinase?
Erk
What type of molecule is Elk?
A transcription factor
How does Raf activate Mek, and Mek activate Erk?
Phosphorylation
In lymphocyte signalling, what is the result of the following pathways:
• MAPK
• PLC-γ & calcineurin
• PLC-γ & PKC
MAPK: AP-1
PLC-γ & calcineurin: NFAT
PLC-γ & PKC: NF-κB
Which molecule activates Lck?
CD45
By removing the phosphate from the inhibitory tyrosine
Which molecule inhibits Lck?
Csk
By phosphorylating the inhibitory tyrosine
Which cytokine is produced through NFκB signalling?
IL-2
